Pathogenesis of Keratin-Containing Inclusions in Liver Disease

描述（由申请人提供）：我们研究的总体目标是了解消化器官中角蛋白中间丝（IF）细胞骨架蛋白的调节、功能和疾病相关性。角蛋白8和18（K8/K18）是肝细胞的IF，其主要功能是细胞保护免受机械和非机械应力（例如细胞凋亡）的影响。这种功能与特定的K8和K18遗传变异易导致肝脏疾病进展的发现一致。K8和K18也是发现与一些肝脏疾病相关的马洛里体（MB）包涵体的主要成分。几个转基因动物研究表明，K8和K8-大于K18的蛋白质比例，加上药物损伤都是必要的，以形成MB。一旦最初形成，MB可以迅速重新诱导，但这种快速倾向于重新形成的机制尚不清楚。我们的建议包括4个目的，以研究MBs的发病机制。前三个目标测试的假设，在特定的氨基酸角蛋白转酰胺是必不可少的MB的形成。第四个目的是检验在MB（再）形成过程中伴侣功能改变的假设。拟议的研究利用细胞培养和小鼠MB模型，以帮助了解其发病机制，并最终了解其在人类疾病中的重要性。4个目的是：（i）使用建立的体内模型研究转氨酶-2（TG 2）缺失和对照小鼠中的MB形成。这一目标是基于我们的初步发现，即TG 2-null小鼠形成MB的能力明显减弱。(ii)在体内/体外鉴定角蛋白转酰胺位点及其交联伴侣，并研究其在细胞培养中交联和包涵体形成中的作用。这是基于以下发现：K8/K18在体外是优异的TG 2底物（K8>K18），（iii）产生表达转酰胺基突变角蛋白的转基因小鼠，并测试突变对MB形成和对肝损伤的易感性的影响，（iv）测试MB诱导损伤对伴侣蛋白功能的影响和伴侣蛋白作为与MB再积累相关的“记忆蛋白”的潜在作用。这是基于我们最近的发现，MB的形成与分子伴侣功能障碍。我们提出的研究可能会产生重要的新的生物学和临床相关信息的发病机制MB。我们预计，该项目的完成将揭示特定的角蛋白氨基酸，使MB的形成和地址，如果MB是旁观者，保护者或促进者的伤害。我们的研究结果也应该阐明伴侣功能障碍是否可以提供“分子记忆”，并因此有助于快速MB的重组。我们的建议代表了一种直接的方法，以充分了解的发病机制和意义的MB，首先在1911年由博士弗兰克马洛里，也可能影响其他夹杂物，发现与其他几个神经和神经肌肉人类疾病的发病机制是未知的。

DESCRIPTION (provided by applicant): The overall goal of our studies is to understand the regulation, function, and disease association of the keratin intermediate filament (IF) cytoskeletal proteins in digestive organs. Keratins 8 and 18 (K8/K18) are the IFs of hepatocytes and their major function is cytoprotection from mechanical and nonmechanical stresses such as apoptosis. This function is consistent with the finding that specific K8 and K18 genetic variants predispose to liver disease progression. K8 and K18 are also the major constituents of Mallory body (MB) inclusions that are found in association with some liver diseases. Several transgenic animal studies demonstrated that K8 and a K8-greater-than-K18 protein ratio, coupled with a drug insult are all essential in order for MBs to form. MBs can be rapidly re-induced once initially formed but the mechanism of such rapid predisposition to reformation is unknown. Our proposal includes 4 aims to examine the pathogenesis of MBs. The first three aims test the hypothesis that keratin transamidation at specific amino acids is essential for MB formation. The 4th aim tests the hypothesis that chaperone function is altered during MB (re)formation. The proposed studies utilize cell culture and mouse MB models to help understand their pathogenesis and, ultimately, their importance in human disease. The 4 aims are: (i) Study MB formation in transglutaminase-2 (TG2) null and control mice using established in vivo models. This aim is based on our preliminary findings that TG2-null mice have a markedly blunted ability to form MBs. (ii) Identify keratin transamidation sites and their crosslinked partners in vivo/vitro and study their role in crosslinking and inclusion body formation in cell culture. This is based on the findings that K8/K18 are excellent TG2 substrates (K8>K18) in vitro, (iii) Generate transgenic mice that express transamidation-mutant keratins and test the effect of the mutations on MB formation and susceptibility to liver injury, (iv) Test the effect of MB-inducing injury on chaperone function and the potential role of chaperones as "memory proteins" in association with MB re-accumulation. This is based on our recent findings that MB formation correlates with chaperone dysfunction. Our proposed studies are likely to generate important new biologic and clinically relevant information regarding the pathogenesis of MBs. We anticipate that completion of this project will shed light on the specific keratin amino acids that allow MB formation and address if MBs are bystanders, protectors or promoters of injury. Our findings should also shed light on whether chaperone dysfunction can provide 'molecular memory" and as such contribute to rapid MB reformation. Our proposal represents a direct approach to fully understand the pathogenesis and significance of MBs that were first described in 1911 by Dr. Frank Mallory, and may also impact on other inclusions that are found in association with several other neurological and neuromuscular human diseases whose pathogenesis is unknown.